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ABSTRACT: LPS stimulation of monocytes/macrophages induces the expression of genes encoding proinflammatory cytokines and the procoagulant protein, tissue factor. Induction of these genes is mediated by various signaling pathways, including mitogen-activated protein kinases, and several transcription factors, including Egr-1, AP-1, ATF-2, and NF-kappaB. We used a genetic approach to determine the role of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway in the regulation of LPS signaling and gene expression in isolated macrophages and in mice. The PI3K-Akt pathway is negatively regulated by the phosphatase and tensin homologue (PTEN). We used peritoneal exudate cells from Pik3r1-deficient mice, which lack the p85alpha regulatory subunit of PI3K and have reduced PI3K activity, and peritoneal macrophages from PTEN(flox/flox)/LysMCre mice (PTEN(-/-)), which have increased Akt activity. Analysis of LPS signaling in Pik3r1(-/-) and PTEN(-/-) cells indicated that the PI3K-Akt pathway inhibited activation of the ERK1/2, JNK1/2, and p38 mitogen-activated protein kinases and reduced the levels of nuclear Egr-1 protein and phosphorylated ATF-2. Modulating the PI3K-Akt pathway did not affect LPS-induced degradation of IkappaBalpha or NF-kappaB nuclear translocation. LPS induction of TNF-alpha, IL-6, and tissue factor gene expression was increased in Pik3r1(-/-) peritoneal exudate cells and decreased in PTEN(-/-) peritoneal macrophages compared with wild-type (WT) cells. Furthermore, LPS-induced inflammation and coagulation were enhanced in WT mice containing Pik3r1(-/-) bone marrow compared with WT mice containing WT bone marrow and in mice lacking the p85alpha subunit in all cells. Taken together, our results indicate that the PI3K-Akt pathway negatively regulates LPS signaling and gene expression in monocytes/macrophages.
The Journal of Immunology 04/2008; 180(6):4218-26. · 5.79 Impact Factor
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ABSTRACT: Cancer patients have an increased risk of thrombosis. Tissue factor (TF) antigen and TF activity associated with microparticles in plasma are elevated in patients with various types of cancer. Of these two measurements, TF activity is considered superior to TF antigen levels because the activity more closely reflects the ability of TF to initiate coagulation. Recent studies showed that platelets also express TF.
To determine the level of TF activity associated with a combined platelet and microparticle sample from cancer patients (n = 20) and healthy individuals (n = 23).
TF activity was measured using a two step chromogenic assay and soluble P-selectin was measured by ELISA in healthy controls and metastatic cancer patients.
We determined the composition of a combined platelet and microparticle sample. The sample consisted of platelets, large microparticles (30-200 nm) and membrane debris. We compared the TF activity of a combined platelet and microparticle sample from cancer patients with that from healthy individuals. We found that TF activity in a combined platelet and microparticle sample from cancer patients was higher than in samples from healthy individuals (21.5+/-12.3 pM (n = 20) versus 8.6+/-6.8 pM (n = 23), mean+/-SD, p < 0.001). Cancer patients also had a higher level of soluble P-selectin compared with controls (18.9+/-5.5 ng/mL versus 13.2+/-2.3 ng/mL, p < 0.001).
This study indicates that measurement of TF activity in a combined platelet and microparticle sample can be used as a simple assay to determine the level of circulating TF.
Thrombosis Research 02/2008; 122(5):604-9. · 2.44 Impact Factor
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James P Luyendyk,
J Daniel Piper,
Michael Tencati,
K Veera Reddy, Todd Holscher,
Rong Zhang,
Jayraz Luchoomun,
Xilin Chen,
Wang Min,
Charles Kunsch,
Nigel Mackman
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ABSTRACT: Oxidative stress contributes to the pathogenesis of many diseases, including atherosclerosis and sepsis. We have previously described a novel class of therapeutic compounds with antioxidant and antiinflammatory properties. However, at present, the intracellular targets of these compounds have not been identified. The purpose of this study was to elucidate the mechanism by which 2 structurally-related antioxidants (AGI-1067 and AGI-1095) inhibit LPS induction of tissue factor (TF) expression in human monocytic cells and endothelial cells.
We found that succinobucol (AGI-1067) and AGI-1095 inhibited LPS induction of TF expression in both monocytic cells and endothelial cells. These compounds also reduced LPS induction of nuclear AP-1 and expression of Egr-1 without affecting nuclear translocation of NF-kappaB. Importantly, these antioxidants inhibited LPS activation of the redox-sensitive kinase, apoptosis signal-regulating kinase-1 (ASK1) and the mitogen-activated protein kinases (MAPKs) p38, ERK1/2, and JNK1/2.
AGI-1067 and AGI-1095 inhibit TF gene expression in both monocytic cells and endothelial cells through a mechanism that involves the inhibition of the redox-sensitive MAP3K, ASK1. These compounds selectively reduce the activation/induction of MAPK, AP-1, and Egr-1 without affecting NF-kappaB nuclear translocation.
Arteriosclerosis Thrombosis and Vascular Biology 09/2007; 27(8):1857-63. · 6.37 Impact Factor
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ABSTRACT: Fondaparinux is a synthetic pentasaccharide that selectively inhibits factor Xa (FXa) in an antithrombin-dependent fashion. This newly developed anticoagulant is used in the prevention and treatment of venous thromboembolism. Recently, we showed that fondaparinux reduces inflammation and protects the kidney from ischemia-reperfusion (I/R) injury. However, the relative contributions of the anticoagulant and anti-inflammatory activities of fondaparinux to the observed protection is unknown. To address this, we chemically modified fondaparinux to abolish its affinity for antithrombin and analyzed the effect of this non-anticoagulant (NAC)-pentasaccharide on binding of U937 cells to P-selectin in vitro and on inflammation in a murine model of kidney I/R injury. NAC-pentasaccharide was as effective as fondaparinux at inhibiting the binding of U937 cells to P-selectin. In addition, NAC-pentasaccharide significantly reduced IL-6 and MIP-2 expression and injury in the kidney I/R model. These findings indicate that the anti-inflammatory activity of fondaparinux can be dissociated from its anticoagulant activity and that NAC-pentasaccharide is protective in kidney I/R injury.
Thrombosis and Haemostasis 01/2007; 96(6):802-6. · 5.04 Impact Factor
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ABSTRACT: Inactivation of the murine tissue factor (TF) gene or tissue factor pathway inhibitor 1 (TFPI) gene results in embryonic lethality, indicating that both are required for embryonic development. We have shown that expression of low levels of TF from a transgene (hTF) rescues TF-null embryos. However, low-TF mice (mTF(-/-)/hTF+) have hemostatic defects in the uterus, placenta, heart, and lung. In this study, we hypothesized that the death of TFPI-/- embryos was due to unregulated TF/FVIIa activity and that the hemostatic defects in low-TF mice were due to insufficient TF expression. Therefore, we attempted to rescue TFPI-/- embryos by reducing TF expression, and to restore hemostasis in low-TF mice by abolishing TFPI expression. Intercrossing TFPI(+/-)/mTF(+/-)/hTF+/- mice generated close to the expected number of TFPI(-/-)/low-TF mice at weaning age from 128 offspring, indicating rescue of TFPI-/- embryos from embryonic lethality. Conversely, a decrease in TFPI levels dose-dependently prolonged the survival of low-TF mice and rescued the hemorrhagic defects in the lung and placenta but not in the heart or uterus. These results indicate that the correct balance between TF and TFPI in different organs is required to maintain hemostasis during embryonic development and in adult mice.
Blood 05/2005; 105(7):2777-82. · 9.90 Impact Factor
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ABSTRACT: Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the down-stream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.
Blood 03/2004; 103(4):1342-7. · 9.90 Impact Factor
